The present invention relates to an electroporation device and its use for facilitating the introduction of a macromolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device (“EKD”) which provides a series of programmable constant-current pulse patterns between electrodes in an array, user control and input of the pulse parameters, and storage and acquisition of data. The electroporation device also comprises a replaceable, or exchangeable, electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk.
Plasmid transfer technology has traditionally been limited in scope because in vivo expression levels resulting from the naked DNA transfer have been low, only fractions of that achieved by viral gene transfer. Numerous investigators have outlined the safety and toxicological concerns with injecting viruses as DNA vectors into animals and humans (Pilaro and Serabian, 1999). Consequently, direct injection of plasmid DNA has become more attractive as a viable alternative. Persistent plasmid DNA transfer is accomplished with the application of a series of electric pulses to drive the DNA into a stable, non-dividing, population of cells. Skeletal muscle cells have provided an ideal target for direct plasmid transfer for DNA vaccines and other applications (Mor and Eliza, 2001; Stoll and Calos, 2002). Enhancement of plasmid delivery using electroporation allows the injected muscle to be used as a bioreactor for the persistent production and secretion of proteins into the blood stream. The expression levels are increased by as much as two to three orders of magnitude over plasmid injection alone, to levels comparable to those of adenoviral-mediated gene delivery and may in some cases reach physiological ranges.
The method of plasmid delivery in vivo, termed electroporation, electro-permeabilization, or electrokinetic enhancement, is simple, efficient and reproducible. It has become valuable for basic research, with great potential for gene transfer and DNA vaccination. Electroporation has been used very successfully to transfect tumor cells after injection of plasmid or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors in humans. Electroporation has been extensively used in mice, rats, dogs and pigs to deliver therapeutic genes that encode for a variety of hormones, cytokines, enzymes or antigens. The numerous tissues and organs that have been targeted include liver, skin, eye, testis, cardiac muscle, smooth muscle, tumors at different locations, and skeletal muscle.
Broadly, electroporation is the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. These pores are commonly called “electropores.” Their presence allows macromolecules, ions, and water to pass from one side of the membrane to the other. Thus, electroporation has been used to introduce drugs, DNA or other molecules into multi-cellular tissues, and may prove to be effective for the treatment of certain diseases. However, the use of electroporation in living organisms has several problems, including cell death that results from generated heat and the inability of electropores to reseal. The beneficial effects of the drug or macromolecule are extremely limited with prior art electroporation methods where excessive cell heating and cell death occurs.
To better understand the process of electroporation, it is important to look at some simple equations. When a potential difference (voltage) is applied across the electrodes implanted in a tissue, it generates an electric field (“E”), which is the applied voltage (“V”) divided by the distance (“d”) between the electrodes.E=V/d 
The electric field intensity E has been a very important value in prior art when formulating electroporation protocols for the delivery of a drug or macromolecule into the cell of the subject. The field intensity is inversely proportional to the distance between the electrodes in that given a voltage, the field strength increases as the distance between the electrodes is decreased. However, a caveat is that an electric field can be generated in a tissue with insulated electrodes (i.e. flow of ions is not necessary to create an electric field). Although not wanting to be bound by theory, it is the flow of ions that opens the electropores and allows movement of molecules into the cells of a subject during electroporation. The flow of electric charge in a conductor or medium between two points having a difference in potential is called the current. The current between electrodes is achieved by the ions or charged particles in the tissues, which can vary among tissues and patients. Furthermore, the flow of conducting ions in the tissue can change between electrodes from the beginning of the electric pulse to the end of the electric pulse.
When tissues have a small proportion of conducting ions, resistance is increased, heat is generated and cells are killed. Ohm's law expresses the relationship between current (“I”), voltage (“V”), and resistance (“R”):R=V/I 
The resistance in the tissue between two electrodes varies depending on the charged particles present therein. Thus, the resistance in the tissue changes from the beginning of the electric pulse to the end of the electric pulse.
Heating is the product of the inter-electrode impedance (i.e. combination of resistance and reactance and is measured in ohms), and is proportional to the product of the current, voltage and pulse duration. Heating can also be expressed as the square of the current, and pulse duration (“t”, time). For example, during electroporation the heating or power (“W”, watts) generated in the supporting tissue can be represented by the following equation:W=I2Rt 
Broadly, prior art teaches that metallic electrodes are placed in contact with tissues and short pulses of predetermined voltages are imposed on the electrodes initiating the cells to transiently open membrane pores. The protocols currently described for electroporation are defined in terms of the resulting field intensities E, which are dependent on short pulses of voltage proportional to the distance between the electrodes, and regardless of current. Accordingly, the resistance or heating cannot be determined for the electroporated tissue, which leads to varied success with different pulsed voltage electroporation protocols. Certainly, the difference in upper limit amplitudes of a voltage pulse between electroporation protocols that facilitate effective electroporation and electroporation protocols that cause the cells to die are very small. Additionally, a definite correlation has been observed between death of cells and the heating of cells caused by the upper limit amplitudes of the short voltage pulses. Thus, the over heating of cells between across electrodes serves as a principal cause for the ineffectiveness of any given electroporation voltage pulsing protocol. Furthermore, the current between electrodes serves as a primary determinant of the effectiveness of any given pulsing protocol, not the voltage across the electrodes.
When electricity is delivered to the cells of a subject, the dose of electricity can be accurately described in terms of charge (“Q”), which is the current (“I”) and the time (“t”), according to the formula:Q=It 
If the current is not constant, as is the case in prior art electroporators, Q represents the time integral of I. In this respect, charged particles, be they ions or molecules, behave in a similar fashion. For example, when silver ions are deposited on an electrode to define the standard unit of electrical charge (the coulomb), only the charge, as defined above, is of importance. A certain minimum voltage must be present to generate a current, but the quantity of ions deposited can not be determined from a pre-determined voltage. Correspondingly, the quantity of charged particles delivered to cells in an electroporator can not be derived from the voltage imposed on the electrodes.
Although electroporation is widely used for laboratory gene transfection and gaining increased importance for non-viral gene therapy, it is generally employed using trial-and-error optimization schemes for lack of methods to predict electroporation's effects on cells (Canatella and Prausnitz, 2001). For example, it has been shown that the efficiency of plasmid gene transfer to skeletal muscle can be significantly improved by the application of an electrical field to the muscle following injection of plasmid DNA. However, this electrotransfer is associated with significant muscle damage that may result in substantial loss of transfected muscle fibers (McMahon et al., 2001). The reduction of the voltage used in the technique can result in a decrease in muscle damage, with a concomitant reduction in expression, but without a significant decrease in the number of transfected fibers.
The effectiveness of electroporation is limited by the fact that there is a threshold value for the pulse intensity below which electroporation does not occur, and an upper limit above which the cells are destroyed. Experimental evidence shows that the difference between the upper and lower limits is so small that it is very difficult to design effective pulsing protocols without undue experimentation. This makes use of the technique difficult.
References in the art directed toward an electroporation apparatus illustrate the usefulness of both an electrode apparatus and an in vivo method of electroporation. Correspondingly there are many U.S. Patents that claim either specific electrodes, or methods for electroporation. For example, U.S. Pat. No. 6,302,874 is a method and apparatus for electrically assisted topical delivery of agents for cosmetic applications; U.S. Pat. No. 5,676,646; is a flow through electroporation apparatus for implanting molecules into living blood cells of a patient; U.S. Pat. Nos. 6,241,701 & 6,233,482 describe a method and apparatus for electroporation mediated delivery of drugs and genes. More specifically, they describe a method and apparatus for electroporation therapy (“EPT”) for treating tumors with a combination of electroporation using the apparatus of the invention and a chemotherapeutic agent to produce regression of tumors in vivo; U.S. Pat. No. 6,216,034 describes a method of programming an array of needle electrodes for electroporation therapy of tissue; U.S. Pat. No. 6,208,893 describes an electroporation apparatus with a connective electrode template; U.S. Pat. No. 6,192,270 describes an electrode assembly for an apparatus and a method of trans-surface molecular delivery; U.S. Pat. No. 6,181,964 describes a minimally invasive apparatus and method to electroporate drugs and genes into tissue. Using EPT as described in the invention, tumors treated by a combination of electroporation using the apparatus of the invention and a chemotherapeutic agent caused regression of tumors in vivo. U.S. Pat. No. 6,150,148 describes an electroporation apparatus for control of temperature during the process, by generating and applying an electric field according to a user-specified pulsing and temperature profile scheme; U.S. Pat. No. 6,120,493 describes a method for the introduction of therapeutic agents utilizing an electric field electroporation apparatus; U.S. Pat. No. 6,096,020 describes an electroporation method and apparatus for generating and applying an electric field according to a user-specified pulsing scheme; U.S. Pat. No. 6,068,650 describes a method of selectively applying needle array configurations for in vivo electroporation therapy; and U.S. Pat. No. 5,702,359 describes an electrode apparatus for the application of electroporation to a portion of the body of a patient with a sensing element for sensing a distance between the electrodes and generating a distance signal proportionate to the distance between said electrodes, and means responsive to said distance signal for applying pulses of high amplitude electric signal to the electrodes proportionate to the distance between said electrodes. All of these cited patents are hereby incorporated by reference.
Significant progress in the enhancement of plasmid expression in vivo and the achievement of physiological levels of a secreted protein has been recently obtained using electroporation (Draghia-Akli et al., 2002). Studies show that injection of a plasmid that expresses growth hormone releasing hormone (“GHRH”), followed by electroporation, is scalable and represents a promising approach for stably producing secreted proteins for treating large mammals (Draghia-Akli et al., 2003a; Draghia-Akli et al., 2003b). Despite the recent advances in naked plasmid delivery (Dean et al., 2003; Fattori et al., 2002), additional improvements in electroporation techniques are needed.
Previous investigators have utilized electroporation devices for plasmid DNA transfer, all of which are conceptually based on constant voltage systems, utilizing a predetermined voltage between the electrodes. Because the impedance between electrodes that are embedded in a tissue can vary from case-to-case, or tissue-to-tissue, a predetermined voltage does not produce a predetermined current. A predetermined voltage pulse causes an unregulated increase in the current flowing through a muscle tissue during the duration of the pulse in addition to the loss of the perfect square wave function. By contrast, a constant-current source actually maintains a square wave function constant current through muscle tissue. However, the existing commercial electroporation devices do not have the firmware design to enable measurement of the exact amount of current to which the cells are exposed. The unregulated current generated with conventional electroporation devices may generate amounts of heat in tissues that can easily kill cells (Martin et al., 2002; Pliquett et al., 2002). For example, a typical electronic 50 milliseconds (ms) pulse with an average current of 5 Amperes (A, or Amp) across a typical load impedance of 25 ohms (Ω) can theoretically raise the temperature in tissue 7.5° C., which is enough to kill cells. The physics of tissue injury caused by electrical shock is reviewed by Lee et al. (Lee et al., 2000). By contrast, the power dissipation is less in a constant-current system and controls heating of a tissue, which may reduce tissue damage and contribute to the overall success of the procedure. Thus, there is a need to avoid the technological problems associated with constant voltage electroporation by providing a means to control effectively the amount of electricity delivered to the cells and thereby achieve proficient electroporation.
The difficulties present in prior-art electrodes stem from the fact that the pulse energy is concentrated in the center of the array, the point where the material to be transfected is deposited. As a result, the spatial distribution of energy delivery assumes a very non-uniform character. Therefore, only a fraction of the cells in the volume encompassed by the electrode assembly is electroporated. Thus, there is also a need to provide a means to effectively control the dosage of electricity delivered to the cells in the inter-electrode space by precisely controlling the ionic flux that impinges on the conduits in the cell membranes.
Furthermore, commercially available electroporation devices and needle arrays typically do not permit injection and electroporation in one combined operation. With these instruments, the procedure requires that the injection needle be inserted into the target muscle for plasmid delivery, and then removed. Subsequently, the electrodes are inserted into the muscle in the proximity of the injected area, usually identified by a skin tattoo. However, the underlying muscle may more or contract so the injection site may not be completely circumscribed by the needle electrodes. Thus, there is a need for an electroporation device that permits injection and electroporation in one combined operation so that the needle electrodes delineate the injection area during the entire electroporation procedure.
In addition, electroporation devices which use skin and muscle invasive replaceable needle arrays as electrodes to deliver the electric current require maintenance of sterile conditions when the needle array replacement occurs. This is necessary from both a medical practice and regulatory compliance viewpoint. Typically, if there is an orifice in the electroporator handle and electrode disk through which the injection needle must pass to deliver solutions to the tissue, the orifice is not sterile. Depending on the skill of the operator, the injection needle may or may not touch the non-sterile surfaces of the orifice. Furthermore, replacement of the electrode disk is typically done manually, risking contamination of the needle array. Thus, there is also a need to provide an electrode disk that allows delivery of the medicinal solution and replacement of the needle array under sterile conditions.